DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates antitumor immunity

Diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG) signaling by converting DAG to phosphatidic acid, thereby suppressing pathways downstream of T cell receptor signaling. Using a dual DGKα/ζ inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1high and TRP1low), and altered peptide ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced more effector cytokines in the presence of cognate antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of tumor cells with low antigen load required antigen recognition, was mediated by interferon-γ, and augmented by DGKi. Adoptive T cell transfer into mice bearing pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi and antiprogrammed cell death protein 1 (PD-1), with increased expansion of low-affinity T cells and increased cytokine production observed in tumors of treated mice. Collectively, our findings highlight DGKα/ζ as therapeutic targets for augmenting tumor-specific CD8 T cell function.


Fig. S2 .
Fig. S2.Representative flow plot for T cell priming and proliferation analysis.General gating scheme for CellTraceViolet-labeled (A-B) TRP1 high or (C-D) TRP1 low CD8 T cells activated in the presence of B cells pulsed with 50pg/ml native Trp1 peptides +/-150 nM DGKi.

Fig. S3 .
Fig. S3.IL-2 augments DGKi-mediated proliferation.CTV-labeled (A) TRP1 high or (C) TRP1 low CD8 T cells were activated with B cells and native Trp1 peptide for 84 hours +/-100U/mL hIL-2 and/or 350nM DGKi.Representative flow plots for (B) TRP1 high or (D) TRP1 low cultured with B cells in the absence of peptide, 350nM DGKi, and denoted concentrations of hIL-2 are shown.Error bars represent SEM (n = 4).Statistical comparisons were conducted within each peptide concentration with a two-way ANOVA and Sidak's test for multiple comparisons.

Fig. S4 .
Fig. S4.DGKi upregulates cytokine transcript and protein expression during T cell priming.(A) TRP1 high CD8 T cells were cocultured with B cells pulsed with 500 pg/ml native Trp1 peptide and proliferation was determined via CTV staining at indicated time points.(B) Cells were also collected for RNA analysis via qPCR and (C) supernatants were collected to determine IL2, IFNγ, and TNFα proteins levels via ELISA at indicated time points (n = 3 for each treatment and time point).Error bars represent SEM.Transcript and protein levels were compared with Two-way ANOVA and Sidak's test for multiple comparisons.Statistically significant P-values are denoted above compared groups.

Fig. S5 .
Fig. S5.DGKi upregulates Myc expression in TRP1 high CD8 T cells in a temporal manner.CTV-labeled TRP1 high CD8 T cells expressing Myc-GFP were activated with 1μg/ml αCD3/28 coated plates for 24, 48, and 72 hours.(A) Proliferation Index was quantified via CTV expression.Myc expression was quantified through (B) percentage of CD8 cells expressing Myc and (C) magnitude of Myc quantified via mean fluorescence intensity.Myc MFI was quantified for every division from CTV intensity for (D) vehicle and (E) DGKi treated TRP1 high CD8 T cells (n = 3 for all groups).Error bars represent SD.Statistical comparisons were conducted with a Two-way ANOVA and Dunnett's test for multiple comparisons to the vehicle control group.Statistically significant P-values are denoted above compared groups.

Fig. S6 .
Fig. S6.Tumor growth is unaffected by DGKi in the absence of antigen-specific T cells.B16 zsgreen or C2VTrp1 Tumor cells were incubated with and without TRP1 high CD8 T cells and increasing concentrations of DGKi for 24 and 48 hours.Zsgreen confluency was measured for (A) B16 and (C) C2VTrp1 tumor only wells with the Celigo Imaging Cytometer.TRP1 high CD8 T cells were cocultured with (C) B16 or (D) C2VTrp1 cells for 24 to 48 hours at varying concentrations of DGKi and %cytotoxicity was calculated from zsgreen confluency (n = 4).Error bars represent SEM.Confluency and cytotoxicity was compared with a Two-way ANOVA with Sidák's test for multiple comparisons.Statistically significant P values are denoted between vehicle (0 nM) and DGKi treated conditions.

Fig. S7 .
Fig. S7.DGKi enhances CD8 T cell cytokine transcript levels and protein expression during coculture with tumor cells.(A) TRP1 high CD8 T cells were cocultured with C2VTrp1 tumor cells for 24 hours in the presence or absence of 100nM DGKi.(B) Cells were collected for RNA isolation and analysis.Transcript levels of IL2, IFNγ, and TNFα was determined by qPCR.(C) Supernatants were collected and ELISAs were performed to determine protein levels of the aforementioned three cytokines.Comparisons of cytotoxicity, transcript, and protein levels were quantified with student's t-test.(D) Effector TRP1 high CD8 T cells were activated with 1μg/ml αCD3/28 (+/-100nM DGKi) for 24 hours followed by assessment of IFNγ and IL2 production by intracellular cytokine staining.

Fig. S8 .
Fig. S8.DGKi and αPD1 in combination decreases CD8 T cell PD-1 expression and increases tumor cell PD-L1 expression.TRP1 high and TRP1 low were adoptively transferred into irradiated C57BL/6 mice one day prior to inoculation with C2VTrp1 tumor cells.Tumors were harvested on day 18 and stained for PD-1 and PD-L1 expression.(A) Representative flow gating scheme for determining PD-L1 and PD-1 expression on tumor and CD8 T cells respectively.(B) Percentage of zsgreen+ tumor cells expressing PD-L1 and (C) PDL1 MFI of tumor cells was quantified.(D) Percentage of PD-1+ of CD8+ T cells along with (E) MFI of PD-1-expressing CD8+ T cells was quantified.Statistical comparison was conducted via One-way ANOVA with Dunnett's multiple comparison test.

Fig. S10 .
Fig. S10.Representative flow gating scheme for intracellular cytokine staining of C2VTrp1 tumors.(A) Single cells were gating on CD8 T cells followed by isolating the adoptively transferred TRP1 high and TRP1 low T cells through CD45.2+ staining.The two TRP1 T cells were differentiated by TCRVβ5 expression with TRP1 low positive for Vβ5 expression and negative for TRP1 high T cells.Subsequently, TRP1 high and TRP1 low cells were gated for their expression of IFNγ, IL2, and TNFα.Quantification of cytokine groups are shown in Fig. 7.

Fig. S11 .
Fig. S11.Enhanced cytokine production by antigen-specific T cells in DGKi treated mice was not found in the spleen.(A) Percentage of adoptively transferred CD8 T cells found in the spleen from bearing C2VTrp1 tumors.(B) The ratio of TRP1 low to TRP1 high cells found in the spleen.Frequency of (C) TRP1 high or (D) TRP1 low producing IL2, IFNγ, and TNFα, was determined via intracellular cytokine staining.Errors bars represent SEM.Statistical comparison was conducted via one-way ANOVA and Dunnett's test for multiple comparison.No statistically significant comparisons were found.